This proposal investigates protein structure, stability, folding and activity to better understand how defects in proteins give rise to disease and how a protein's stability and activity may be manipulated. The model system is the extremely well characterized four helix bundle protein, Rop. The applicant will analyze the thermodynamic properties and determine the high resolution crystal structures of a series of variants of Rop with completely repacked hydrophobic cores. These studies will provide insights into the molecular basis of the physical properties displayed by natural proteins and illustrate how these properties can be perturbed. They will also reveal the interactions which specify the orientation of helix-helix associations. Because Rop folds and unfolds particularly slowly, the applicant will use real-time NMR to characterize the nature of folding intermediates in greater structural detail, providing an important contribution to our understanding of the role of intermediates in protein folding. Rop regulates the replication of ColE1 plasmids by interacting with hairpin-pair complexes. The applicant will study the Rop-RNA interaction, with particular emphasis on the identification of mutations that enhance the affinity or alter the specificity of the interaction. These studies will be interpreted in the context of the high resolution structures of Rop, the hairpin pair, and the Rop-hairpin pair complex. In the course of these studies, a range of biochemical, molecular biological and biophysical techniques will be employed, including protein expression and purification, electrophoresis, circular dichroism, fluorescence, NMR, and x-ray crystallography.